Mycotoxin toxicity and its alleviation strategy on female mammalian reproduction and fertility.

There are both pro-oxidants and anti-oxidants in cells to keep a redox homeostasis in a healthy body. Excessive free radicals that exceed the cell scavenging capacity would attack organelles and macromolecule, causing cellular oxidative injury on lipid membranes, DNA, and proteins, which further results in membrane organelle dysfunction and DNA damage [155] . Close association between oxidative stress and a number of reproductive disorders such as endometriosis, polycystic ovary syndrome (PCOS), preeclampsia, and unexplained infertility has been revealed [156] . More precisely, redox system may influence the mammal female reproductive life span, including follicular growth, oocyte meiotic maturation, fertilization, and embryonic development [155] . Oxidative stress is commonly found after mycotoxin exposure. A relatively high level of ROS was seen in mouse oocyte, embryo, blastocyst, and maternal placenta after NIV, HT-2, OTA, or DON exposure [49] , [56] , [59] , [65] , as well as in the fetal ovary upon maternal exposure to HT-2 [73] . Similarly, exposure to FB1 or AFB1 in porcine oocyte [54] , [152] , ZEN in porcine GCs [157] , and AFB1 in bovine embryo all triggers excessive intracellular ROS accumulation [158] . Increased expression of stress-related genes, such as oxidation resistance 1 (OXR1), NADPH-dependent thioredoxin reductase (TRR1), heat shock protein 70 (HSP70) and metallothionein-1/2 genes, was seen in the T-2 toxin exposed placenta of pregnant rat [66] . As is well known, 95 % intracellular free radicals are ROS which are mainly produced via electron leakage in mitochondria respiratory chain [159] . However, mitochondria are quite susceptible to ROS since core gene elements about effective histone protection, oxidant scavenging and DNA damage repair systems are lacking in mitochondrial DNA (mtDNA) [160] , [161] . The ROS-mediated mitochondria damage would aggravate the formation of oxidative products and further result in a vicious cycle, as shown by elevated oxidants and reduced ATP levels which have been discussed before [155] . Furthermore, a significant loss of MMP accompanied by elevated ROS levels was observed in mouse embryo and blastocyst after exposure to HT-2 and OTA respectively [56] , [59] . Similar findings were shown in ZEN-exposed porcine GCs and FB1-exposed porcine oocytes [54] , [157] . These results imply that mitochondrial dysfunction is closely related to imbalanced redox homeostasis after mycotoxin exposure. Of course, there are still limited strategies in cell to prevent or relieve oxidative stress. On the one hand, enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), thioredoxin/ thioredoxin reductase/peroxiredoxin (Trx/TrxR/Prx), and the glutathione/glutathione peroxidase (GSH/GPx) systems help to degrade peroxides or inhibit ROS production [155] , [162] . Nearly 50 % decreased expression of CAT, SOD2 and GSH-PX was seen in both FB1-exposed porcine oocyte and ZEN-exposed rat placenta [54] , [68] . Whereas, significantly increased expression of SOD and CAT was seen when mice were in utero exposed to HT-2 [73] , suggesting a compensatory mechanism in cell to prevent oxidative damage. On the other hand, damaged proteins, DNA, and organelles caused by ROS may activate autophagy pathways to deliver them to the lysosome for degradation and recycle [155] ( Fig. 3 ). In accordance with excessive ROS generation, aggregated location of lysosomes in the cytoplasm was found in ZEN-, AFB1-, and FB1-exposed oocytes [115] , [116] , [117] . Besides, the expression level of autophagy-related genes (ATGs) and autophagy biomarkers, such as LC3, Beclin1, and p62 increased in porcine oocyte or blastocysts after AFB1 exposure, as well as in the ovaries of offspring after ZEN maternal exposure [120] , [147] , [152] . In the offspring ovaries, maternal ZEN exposure also led to reduced p-mTOR/S6K1, a negative regulator of autophagy [120] . Moreover, decreased autolysosomes but increased autophagosomes were found in ZEN-exposed gilt primary endometrial cells, demonstrating that ZEN not only induces autophagy but also suppresses autophagy flow [163] . These findings confirm that autophagy is also quite activated to resist oxidative damage caused by mycotoxin. However, in worse conditions, cell viability would be compromised upon severe oxidative damage, leading to cellular apoptosis or death. Apoptotic signals, shrunken and irregularly shaped or degraded chromatin were seen in embryo and oocyte of swine and rat GCs after AFB1 and T-2 exposure [147] , [152] , [164] , indicating an obvious cytotoxicity of mycotoxin on cell proliferation and viability. As shown in the Fig. 3 , the mitochondria-dependent apoptosis pathway is a major intracellular apoptotic signaling pathway characterized by MMP depolarization and the release of cytochrome c from the mitochondria into the cytoplasm, which further promotes the activation of a downstream caspase cascade [165] . Mitochondrial dysfunction was commonly found after mycotoxin exposure, which has been discussed before. The co-localization level of mitochondria and cytochrome c was decreased in AFB1 exposure groups [147] , implying that AFB1 exposure caused apoptosis via the release of cytochrome c . BCL-2 family members control apoptotic progression and can be separated into two groups: pro-survival members, such as BCL2, BCL2L1, and BCL2L2, are crucial for cell viability protection; whereas BAX and BAK act as pro-apoptotic proteins involved in cell death [166] . About 5-fold increase in BAX and 4-fold increase in BCL-2 led to a much higher BAX/BCL-2 ratio in the 100 nM T-2 toxin-exposed rat GCs compared with that in the control groups [164] . Similar findings were reported when oocyte or embryo of ewes and swine were exposed to AFB1 [58] , [147] , [152] . Besides, ZEN and T-2 toxin-exposed GCs showed an increase in caspase-9 and caspase-3 activity, which was alleviated when cells were pretreated with caspase inhibitor Ac-LEHD-cho, VAD-FMK or Ac-LEHD-FMK [157] , [164] . Consistent with this, inhibited apoptosis was seen in mouse oocyte pretreated with caspase-3 inhibitor Ac-DEVD-cho or si-RNA mediated p21 or p53 knockdown before OTA exposure [167] . Tumor suppressor p53 protein has been known to contribute to genomic integrity protection and act as a transcriptional factor to induce BCL-2 family members gene expression [168] . Similarly, p21 can stimulate the expression of apoptosis-related genes such as E2F family, NF-κB, c-Myc, and STAT [169] , [170] . Mitochondria-mediated apoptotic pathways involving caspases, p53, and p21 appear to be a significant mechanism underlying apoptosis following mycotoxin exposure. And pretreatment with antioxidant Trolox significantly inhibited ROS generation and reduced apoptosis rate compared with T-2 toxin-exposed GCs [164] . Collectively, we propose that ROS-mediated oxidative damage to cells causes mitochondrial dysfunction, subsequently inducing autophagy and leading to caspases-, p53-, and p21-dependent apoptotic pathways, which serve as a major mechanism of impaired cell developmental potential. In addition to classic apoptosis signaling pathway, phosphatidylinositide3-kinases-protein kinase B (PI3K-AKT) signaling pathway also seems to be closely related to apoptosis induced by mycotoxin. PI3Ks are a kind of lipid kinases which could reversibly phosphorylate phosphatidylinositol (PI) to generate phosphoinositides (PIs), while phosphatases and tensin homolog (PTEN) could dephosphorylate PIP2 to PIP3. The most studied PI3Ks consist of a regulatory subunit p85 encoded by Pik3r1 , Pik3r2 , Pik3r3 , and a catalytic subunit p110 encoded by Pik3ca , Pik3cb , Pik3cd [171] . The conversion of PIP2 to PIP3 promotes activation of AKT. In line with these facts, there was a considerably increased expression of Pik3r1 , Pik3r5 , and Pten but reduced AKT1/2 following OTA or ZEN exposure to porcine GCs [45] , [172] . Based on single-cell transcriptome analysis, crosstalk between AKT pathway and TNF signaling in porcine and mouse GCs exposed to ZEN was observed, which was further confirmed by increased resistance to ZEN-induced apoptosis and PI3K-AKT pathway activity after TNF-α silencing [173] . Moreover, increased apoptosis with inhibited PI3K-AKT activity of porcine GCs was seen after knockdown of the ITGA7, a member of integrin alpha chain family, while it was attenuated after up-regulation of ITGA7 expression by cyanidin-3-O-glucoside supplement [172] . Consistent with this, ITGA7 exerted anti-apoptotic effects, showing reduced cytochrome c levels in cytoplasm and inhibited caspase-9 and caspase-3 cleavage in oesophageal squamous cell carcinoma [174] . These facts suggest that TNF/TNFR and ITGA7 may act upstream to regulate PI3K-AKT activity in mycotoxin-induced apoptotic signaling pathway ( Fig. 3 ).

It is recognized that there are critical windows of susceptibility to genotoxins, and genetic damage in utero may trigger diseases in later life. Uterus histologic changes characterized by metaplasia and hypertrophy of the epithelial cells and hypercellularity of the stroma were shown when pregnant rats were exposed to ZEN [22] . Besides, in vitro exposure to ZEN led to the failure of endometrium decidualization in human endometrial stromal cells [63] . The alterations of uterine structure and aberrant decidualization induced by mycotoxin imply unsuccessful embryo implantation and pregnancy outcomes. Once an embryo is implanted in the maternal uterus, the placenta makes sure that nutrients and oxygen, not harmful compounds, from the mother reach the fetus. In DON-exposed pregnant mice, granule- and vacuole-like changes of trophocytes, and immunological changes were found, as well as significantly reduced junctional proteins such as ZO-1, E-cadherin and claudins [64] , [65] . Consistent with this, in vivo exposure to T-2 and ZEN also resulted in damaged placentas [66] , [67] , [68] . Furthermore, disrupted nutrient transport and lipid metabolism, along with suppression of metabolism-related enzyme expression in the placenta were seen when rats were exposed to ZEN or T-2 in vivo [67] , [68] . In addition, the placenta is not only related to compound transport and metabolic functions, but also an essential endocrine organ for fetal development. Down-regulated gene expression in prostaglandin synthesis was confirmed in the rat placenta by RNA-seq after in vivo exposure to ZEN [68] . Similarly, a nearly 4-fold increase in mRNA and protein levels of corticotrophin-releasing hormone (CRH) was found in human placenta-related JEG-3 cells after 100 nM AFB1 exposure compared with the control groups [69] . These findings show that the placental transport and endocrine signaling pathways are greatly affected by mycotoxin, which may be the reasons for the negative fetal health and survival after mycotoxin exposure during gestation. Indeed, decreases in body weight and crown–rump length and increases in incidence of hydrocephalus, skeletal anomalies, and fetal deaths were found in rats after FB1 exposure [70] . OTA exposure also resulted in a significantly decreased number and litter size of offspring compared with the control groups [50] . Similar findings were recorded when mouse or rabbit were exposed to AFB1, T-2, and DON [64] , [71] , [72] , suggesting the decreased maternal fertility after mycotoxin exposure. In addition to adverse effects on maternal reproduction, early oogenesis defects and perturbed intercellular communication network between oocyte and GCs were shown in the female fetuses when pregnant mice were orally administered with HT-2 and ZEN [73] , [74] . Similarly, inhibition of primordial follicle assembly, follicle atresia, significant increases of multi-oocyte follicles (MOFs) and reduced percentage of resting and growing follicles in F1 rats or gilts ovaries were demonstrated after ZEN and OTA exposure [25] , [75] , [76] , which may lead to premature oocyte depletion in adulthood. These findings confirm that mycotoxin exposure during pregnancy may not only impair maternal health, but also undermine the reproductive potential of offspring by placenta or breast milk transportation ( Table 3 ). Table 3 Mycotoxin toxicity on fetus growth and offspring fertility. Mycotoxin Acronym Models In v itro/vivo Concentration; ways Phenotype/Mechanisms Ref Aflatoxins AFB1 Fetus from maternal exposed sextual mature rabbits In vivo 1 mg/Kg BW at GD 6–18 Reduced fetus weight and developmental toxicity in liver, heart and skeleton [71] Zearalenone ZEN Placenta from exposed rat In vivo 2.5–20 mg/Kg/day BW gavage at GD 14–21 Placenta dysfunction includes affected hormone (LH and FSH) secretion, nutrient transport and lipid metabolism via oxidative stress-related AKT1/ERK1/2/mTOR-mediated autophagy and apoptosis signaling pathway [68] Ovary from the F1 newborn at 0–21 days or 160–190 days after birth of the exposed porcine In vivo 200, 500 and 1000 μg/Kg BW diet exposure during pregnant and lactating days Decreased follicle integrity and increased estrogen receptor mRNA expression, which may lead to a premature exhaustion of follicle pool [76] Uterus from exposed adult rats In vivo 3 mg/Kg gavage for 28 days Alerted uterine structures include metaplasia and hypertrophy of epithelial cells, as well as hypertrophy and hyperplasia of the myocytes [22] Offspring (ovary, uterus, placenta) of the exposed pregnant rat In vivo 5–20 mg/Kg BW at GD 0–21 Growth restriction, hormone secretion disorders, abnormal uterus and ovary structure include thinning uterine layer and follicle atresia through regulations on Esr1, gonadotropin-releasing hormone receptor (GnRHr), ATP binding cassette transporters, and 3β-HSD expression [75] Ovary of fetus from exposed pregnant mice at PND 0 or 3 In vivo 40 μg/mL diet exposure at 16.5 days post coitum Inhibited primordial follicle assembly and disturbed germ cell-granulosa cell communication network via Hippo signaling pathway, as well as abnormal mitochondria activity and HDAC2 expression which further induce autophagy and epigenetic changes [74] , [120] Ovary and oocyte of suckled offspring from exposed pregnant mice at postpartum days 0 and 21, respectively In vivo 20 and 40 μg/Kg BW/day diet exposure at 18.5 days post coitum of pregnant mice Inhibited primordial follicle assembly, reduced follicle numbers, oocyte maturation and development capacity induced by DNA damage, apoptosis via RIG-I-like receptor signaling pathways, and disturbed epigenetic modifications [124] Uterus from exposed pregnant porcine In vivo 1–10 mg/Kg BW at GD 8–14 Highly expressed immune system via drug metabolism-cytochrome P450, the cytokine-cytokine receptor interaction and calcium signaling pathways [204] Fecal and ovarian tissue from neonatal female offspring, and maternal ovary of exposed pregnant mice In vivo Exposure for 21 days Diminished ovarian reserve and granulosa cells apoptosis in maternal ovary and reduced oocyte developmental capacity and changed gut microbiota abundance in the lactating offspring [96] Ochratoxin A OTA Pup from exposed F0 female and male rats In vivo 0.026–1.0 mg/Kg diet exposure at gestation and postnatal days Pup loss, delayed vaginal opening, increased multi-oocyte follicles and abnormal proportion in resting and growing follicles numbers of female F1 [25] Embryo or ovary of offspring from exposed pregnant mice In vivo 0.35–35 μg/mL diet exposure at GD 12.5 to 15.5 or until delivery Oxidative stress related DNA damage, meiosis prophase Ⅰ arrest and apoptosis [134] Fumonisins B FB1 Fetus from exposed pregnant rat In vivo 50 mg/Kg/day BW at GD 3–16 Maternal and foetal toxicity in liver, kidney, and brain [70] Trichothecenes T-2 Placenta and fetus from exposed pregnant rat In vivo 2 mg/Kg BW diet exposure for 1–12 h or 24 h at gestation day 13 Cell apoptosis induced by oxidative stress through MAPK mediated apoptotic pathway [66] , [67] Fetus from different exposed gestation day of pregnant mice In vivo 2 mg/Kg/day BW orally inoculated at GD 8.5–16.5 Developmental toxicities include cell apoptosis in nervous and skeletal system [72] HT-2 Ovary from the fetus in the exposed or normal pregnant mice In vivo and in vitro 10–120 ng/Kg BW diet exposure for 3 days at the gestation day 14.5, while 0.75, 1.5 and 3 nM for 3 days in vitro exposure of ovary at the GD 14.5 Oocyte meiotic prophase Ⅰ defects induced by oxidative stress related mitochondria dysfunction, DNA damage, elevated homologous recombination, and apoptosis [73] DON Placenta from exposed pregnant mice at GD 18.5, while BeWo cells from American Tissue Culture Collection In vivo and in vitro 1–5 mg/Kg/day BW gavage at GD 9.5–11.5 in vivo, while 0–750 nM for 1–24 h in vitro Placenta structural and functional damage induced by oxidative stress-related apoptosis or necrosis via Nrf2/HO-1 pathway [65] Fetal and placenta from the exposed pregnant mice at GD 17 In vivo 6.25–12.5 mg/Kg diet exposure at GD 0–17 Reduced fetal survival induced by placenta damage via imbalanced immune responses [64] Mycotoxin toxicity on fetus growth and offspring fertility.

This work was supported by the National Key Research and Development Program of China (2023YFD1300502), the Fundamental Research Funds for the Central Universities of China (KYT2024002, KJJQ2025001, RENCAI2024011), Natural Science Foundation of Guangxi in China (2021GXNSFDA220001).

The combination of the matured oocyte from follicles and sperm embarks on the journey of developing offspring, following early embryo development and fetus growth. As shown in the Fig. 1 , based on the frequent exposure and complex female reproductive processes, mycotoxin is reported to widely affect the female mammalian reproductive process, including follicular development, oocyte maturation, early embryo development, fetus growth, and offspring fertility, which are just recently clarified. Fig. 1 Mycotoxin exposure affect female mammalian reproduction. Mycotoxin is widely found in moldy corn, wheat and their related products such as bread, as well as in fruits, which could enter the human and animal through diet. Long-term mycotoxin exposure would affect the female reproductive processes, including follicle development, oocyte maturation, zygote formation, early embryo development and fetus growth following implantation and placentation. In addition to the adverse effects on the maternal reproductive potential, the fertility of offspring is also influenced, showing abnormal primordial germ cells proliferation and differentiation, and subsequent reproductive processes similar to those of the mother. Mycotoxin exposure affect female mammalian reproduction. Mycotoxin is widely found in moldy corn, wheat and their related products such as bread, as well as in fruits, which could enter the human and animal through diet. Long-term mycotoxin exposure would affect the female reproductive processes, including follicle development, oocyte maturation, zygote formation, early embryo development and fetus growth following implantation and placentation. In addition to the adverse effects on the maternal reproductive potential, the fertility of offspring is also influenced, showing abnormal primordial germ cells proliferation and differentiation, and subsequent reproductive processes similar to those of the mother.

Due to the unpredictable and unavoidable presence of mycotoxin, its toxic effects especially on reproductive system have been confirmed recently. More than 400 mycotoxins have been recognized, while AFB1, ZEN, OTA, FB1, HT-2, T-2, DON, and NIV are mainly discussed in this review. As shown in the Fig. 2 and Fig. 3 , multiple in vivo and in vitro experiments suggest that mycotoxin exposure induces impairments in maternal follicle assembly, embryo or fetus development, and reproductive potential of offspring, accompanied by HPG or gut-ovarian axis disorders, compromised placenta ABC transporters, disrupted organelle and cytoskeleton dynamics, cell cycle progression failure, abnormal epigenetic modifications, and oxidative stress-induced dysfunction. There are still limited ways for mycotoxin elimination and adverse effects mitigation, including physical, chemical, and biological methods, as well as the use of natural antioxidant compounds, whereas their applications are not widespread. The issues described below should be considered in the future. Firstly, species-specific should be considered in mycotoxin toxic tests. Mice, swine, sheep, rabbits, and cattle are mainly used for mycotoxin toxic tests, and the susceptibility of these model animals has been verified. Similarly, estrogen production in GCs of cattle is inhibited while that in swine is increased when exposed to the ZEN metabolite, α-ZOl [80] , [203] . These findings, together with the facts that different transcription or expression levels of epigenetic modifications-related kinases are not consistent among different models, suggest a possibility of a species-specific response to mycotoxin. Moreover, multiple assessments have revealed the significant threat of mycotoxin exposure to human health via dietary intake and exposure to dust in residences and workplaces, particularly during the conception period. However, most evidence only focuses on toxicological evaluations; the toxic effects and mechanisms of mycotoxin in humans have not been fully explored. Moreover, limited human epidemiological studies have reliably identified the involvement of mycotoxin in reproductive cancer development. Human-derived models may be preferable and encouraged for use in specific research, such as BeWo cells, ex vivo perfused human placental model, and organoid. Secondly, time-specific, including the age of animals and the duration of experimental protocols may also influence the outcomes after mycotoxin exposure. A reduced follicle integrity in F1-newborns but normal oocyte and embryo developmental potential in F1-prebubertal gilts was seen when maternal sow were in vivo exposed to 1 mg/Kg feed ZEN during gestation and lactation [76] . While, administration with 1 mg/Kg BW ZEN during 7–10 gestation day (GD) induced retarded embryo disk development of sexually mature sow [57] . Similarly, pyknotic or karyorrhectic cells of different extents and regions were observed when pregnant mice were orally exposed to 2 mg/Kg BW for 1 day from 8.5 to 15.5 GD respectively [72] . Intestinal barrier-related genes, such as ZO-1 , Occludin , and Claudin , as well as inflammation-associated genes Il-6 , Il-10 , and Tnf , were significantly reduced in postnatal day 21 (PND 21) young mice after pregnant mice were given T-2 toxin from 14 GD to lactation day 21 (LD21), whereas Tnf was increased in PND28, and all of them were markedly elevated in PND 56 which may be due to a self-repair mechanism activation [95] . These findings imply that the physiological state (age) and the time point chosen for animal treatment or detection directly influence the experimental conclusions. Thirdly, dose-specific, especially for immune and anti-oxidative response progression. A range of cytokines, such as IL-1, IL-2, IL-4, IL-6, IL-10, and TNF were up-regulated after the DON or ZEN exposure [64] , [204] . However, 0.05 mg/kg T-2 toxin induced the down-regulated IL-6 and IL-10 in comparison to 0.005 mg/kg groups in the ileum of young mice after maternal mycotoxin exposure [95] , suggesting that the immune stimulation or immunosuppression may rely on the mycotoxin dosage, being activated at low concentrations but inhibited at high doses. Similar explanations for anti-oxidative response system and a complementary mechanism might exist in preventing oxidative damage. Moreover, highly hyperplastic follicles were seen in the ovaries of weaned piglets after maternal exposure to 1.5 mg/Kg ZEN, while 3 mg/Kg ZEN led to numerous follicular atresia [130] . Micromolar OTA reduced cumulus expansion, while nanomolar OTA exposure showed normal cumulus-oocyte complex morpho-functional parameters [205] . Above all, a dose-specific effect of mycotoxin toxicity and related mechanistic research should be considered. Note that extra high exposure levels of mycotoxin may lead to nonspecific toxic effects. Fourthly, mechanism-specific. Multiple researches explain the effects on endocrine system, cell cycle, organelles, cytoskeleton, epigenetic modifications, and redox homeostasis, mostly through alterations in related proteins or mRNA expression, which seem to be common features of mycotoxin toxicities. There is considerable lack of knowledge pertaining to mycotoxin specific functional targets as well as molecular signaling pathways in the reproductive system from a biochemistry perspective. For example, in addition to the analysis of related enzyme levels, there is a need to investigate the possible upstream and downstream factors of aberrant epigenetic modifications. Advancements in single-cell transcriptomics and multi-omics technologies can probably be used to identify relevant target molecules. Moreover, due to the small number of cells in the early embryo and highly dynamic changes in cell differentiation, it is difficult to study the impact of mycotoxin on PGCs assembly and placenta formation in embryonic and extra-embryonic cells respectively. Integration of spatial transcriptomics and advanced imaging techniques will enable the characterization of the spatiotemporal distribution of these dynamic changes in cell fate, and therefore, it may provide insights into altered compositions of these clusters after mycotoxin exposure. These novel approaches will contribute to more precisely explaining the adverse toxic effects and mechanisms previously poorly described. Clarification of the above issues may facilitate a deeper understanding of toxic effects and mechanisms of mycotoxin on mammalian female reproductive process, which could help better eliminate or alleviate mycotoxin toxicities and prevent reproductive damage, both in maternal and offspring. Meanwhile, with in-depth insights into the constant interaction between exposomes that depict exposures during the entire life of an individual [206] , it is essential to identify whether there are additive or synergistic toxic effects on reproduction between mycotoxin and other exposures. Moreover, given the prevalence of mycotoxin in the food/feed and limited ways of elimination, good practices in agriculture and manufacture, together with appropriate environment for storage are important strategies in the first step to prevent mycotoxin contamination. Regulatory thresholds for significant levels of mycotoxin in food/feed set by authorities such as the National Health Commission (NHC) and Ministry of Agriculture and Rural Affairs of China (MARA), the United States Food and Drug Administration (FDA), and the World Health Organization (WHO) should be rigorously implemented for high-quality and safe food/feed [4] . Continuous monitoring of mycotoxin at a systematic level, not only in food/feed products, but also in specific environments such as factories, schools, and household kitchens is favorable for putting forward a newer baseline understanding of the mycotoxin in ecological networks. Biomarkers in body fluids have helped reveal the widespread presence of mycotoxin among the population and its close relationship with adverse reproductive outcomes discussed above. They may provide more accurate exposure assessments and establish the relationship between biomarkers detected in biofluids and mycotoxin intake by mass spectrometry methods, especially when coupled with machine learning algorithms, and help identify high-risk individuals in clinical practice in the future.

The toxic mechanisms of mycotoxin have been widely explored in various cell types. The liver, kidney, and intestine are the major target organs, but each kind of mycotoxin theoretically has its specific primary target due to its different chemical structure. For example, ZEN can bind with estrogen receptor to further activate estrogen-like response signaling pathways since its structure is similar to estrogen [40] , [75] . Thus, the toxicities of ZEN are closely associated with reproduction. The structures of OTA and FBs are similar to amino acid phenylalanine (Phe) and sphingosine respectively, which are related to phe-synthetase activity and sphingolipids biosynthesis [3] , [77] . Moreover, trichothecenes are shown to decrease the activity of peptidyl-transferase, an enzyme involved in protein synthesis, by combining with 60S ribosomal subunit [3] . AFB1 exerts its toxic properties required metabolic activating by CYP enzymes which can be detected in the liver and placenta [78] , implying its potential toxicities on metabolism and reproduction. As shown in the Fig. 2 and Fig. 3 , the specific mechanisms of mycotoxin toxicities on reproduction have been explored recently. And these could be divided into six aspects: endocrine system, placenta ABC transporters, organelle and cytoskeleton dynamics, cell cycle control, genomic stability, and redox homeostasis. Fig. 2 Toxic mechanisms of mycotoxin on endocrine system, placenta ABC transporters, and organelle and cytoskeleton dynamics. Mycotoxin can induce maternal endocrine system disturbance in both the hypothalamic-pituitary–gonadal (HPG) axis and gut-ovarian axis. Gonadotropin-releasing hormone (GnRH), a neuropeptide produced by hypothalamus, promotes the secretion of pituitary gonadotrophins and sex hormones via HPG axis, further activates estrogen response elements (EREs) to stimulate target gene expression for regulating reproductive system. A negative feedback loop also exists in the HPG axis. And kisspeptin, a neuropeptide from hypothalamus, could act as an upstream moderator of GnRH secretion by interacting with its receptor GPR54. Gut-ovarian axis shows that there is crosstalk between microbes in the gut and hormones for host reproduction. Additionally, ABC transporters in the placenta may participate in mycotoxin transport to the fetus and further lead to transplacental toxicity in the offspring. Moreover, the dynamics of organelles such as endoplasmic reticulum, Golgi apparatus, mitochondria and their distribution-related cytoskeleton structure are disrupted by mycotoxin. Microtubule-based spindle formation is inhibited in the oocyte, owing to abnormal activity of regulators of spindle assembly such as γ-tubulin, Aurka, PLK1, and p-MAPK, and an aberrant level of tubulin acetylation related to microtubule stability after mycotoxin exposure. Similarly, mycotoxin exposure results in a loss of mDIA1, Profilin1, and ROCK localization, which further disturbs microfilament functions and ultimately induces oocyte meiotic maturation failure. Fig. 3 Toxic mechanisms of mycotoxin on cell cycle control, genomic stability, and redox homeostasis. Mycotoxin exposure affects the activity of Wee1/Myt1 for Cyclin B1-CDK1 complex (MPF) and DNA damage repair signaling pathway-based cell cycle control. Inadequate Cyclin B1 synthesis and continuous activation of checkpoint proteins such as BUBR1 and MAD2 inhibit oocyte meiosis after mycotoxin exposure. Besides, abnormal expression of histone modification-related kinases such as histone methyltransferases (HMTs), histone demethylases (KDMs), histone acetyl-transferases (HATs), and histone deacetylases (HDACs), together with DNA methyltransferases (DNMTs) which are responsible for DNA methylation caused by mycotoxin, leads to disrupted genomic stability. Moreover, excessive ROS that exceeds the cell scavenging capacity activates the expression of autophagy-related genes (ATGs) and autophagy based on LC3, Beclin1, and p62, while reducing p-mTOR/S6K1 activity, inducing autophagolysosomal formation after mycotoxin exposure. Furthermore, severe ROS-mediated oxidative damage to the cell causes mitochondrial dysfunction, subsequently leading to caspase-, p53-, and p21-dependent apoptotic pathways. Besides, tumor necrosis factor receptor TNFR and transmembrane heterodimer ITGA7 serve as upstream regulators in the PI3K-AKT signaling pathway, modulating mycotoxin-induced apoptosis. Toxic mechanisms of mycotoxin on endocrine system, placenta ABC transporters, and organelle and cytoskeleton dynamics. Mycotoxin can induce maternal endocrine system disturbance in both the hypothalamic-pituitary–gonadal (HPG) axis and gut-ovarian axis. Gonadotropin-releasing hormone (GnRH), a neuropeptide produced by hypothalamus, promotes the secretion of pituitary gonadotrophins and sex hormones via HPG axis, further activates estrogen response elements (EREs) to stimulate target gene expression for regulating reproductive system. A negative feedback loop also exists in the HPG axis. And kisspeptin, a neuropeptide from hypothalamus, could act as an upstream moderator of GnRH secretion by interacting with its receptor GPR54. Gut-ovarian axis shows that there is crosstalk between microbes in the gut and hormones for host reproduction. Additionally, ABC transporters in the placenta may participate in mycotoxin transport to the fetus and further lead to transplacental toxicity in the offspring. Moreover, the dynamics of organelles such as endoplasmic reticulum, Golgi apparatus, mitochondria and their distribution-related cytoskeleton structure are disrupted by mycotoxin. Microtubule-based spindle formation is inhibited in the oocyte, owing to abnormal activity of regulators of spindle assembly such as γ-tubulin, Aurka, PLK1, and p-MAPK, and an aberrant level of tubulin acetylation related to microtubule stability after mycotoxin exposure. Similarly, mycotoxin exposure results in a loss of mDIA1, Profilin1, and ROCK localization, which further disturbs microfilament functions and ultimately induces oocyte meiotic maturation failure. Toxic mechanisms of mycotoxin on cell cycle control, genomic stability, and redox homeostasis. Mycotoxin exposure affects the activity of Wee1/Myt1 for Cyclin B1-CDK1 complex (MPF) and DNA damage repair signaling pathway-based cell cycle control. Inadequate Cyclin B1 synthesis and continuous activation of checkpoint proteins such as BUBR1 and MAD2 inhibit oocyte meiosis after mycotoxin exposure. Besides, abnormal expression of histone modification-related kinases such as histone methyltransferases (HMTs), histone demethylases (KDMs), histone acetyl-transferases (HATs), and histone deacetylases (HDACs), together with DNA methyltransferases (DNMTs) which are responsible for DNA methylation caused by mycotoxin, leads to disrupted genomic stability. Moreover, excessive ROS that exceeds the cell scavenging capacity activates the expression of autophagy-related genes (ATGs) and autophagy based on LC3, Beclin1, and p62, while reducing p-mTOR/S6K1 activity, inducing autophagolysosomal formation after mycotoxin exposure. Furthermore, severe ROS-mediated oxidative damage to the cell causes mitochondrial dysfunction, subsequently leading to caspase-, p53-, and p21-dependent apoptotic pathways. Besides, tumor necrosis factor receptor TNFR and transmembrane heterodimer ITGA7 serve as upstream regulators in the PI3K-AKT signaling pathway, modulating mycotoxin-induced apoptosis.

Due to increasing and unavoidable toxigenic fungi contamination in food and feed worldwide, it poses an enormous threat to human and animal health. The efforts towards mycotoxin elimination mainly focus on screening and breeding crop varieties with resistance traits, mycotoxin degradation using physical, chemical, and biological methods, and protective effects of antioxidant compounds at the individual or cellular level. Rapid bioinformatics development accelerates resistant genotypes and resistance-associated proteins identification. PR10 expression and aFIM silence in maize were helpful in reducing AFB production [175] , [176] . Heating is the traditional physical method for mycotoxin degradation, while it is less cost-effective. Adsorbents (such as nanosilicate platelets, hydrated sodium calcium aluminosilicate, modified hallosite nanotubes) and extraction agents (such as 90 % acetone, 80 % isopropanol and methanol) were developed to minimize mycotoxin toxicity through hydrogen bonding, π-π stacking, electrostatic and hydrophobic interactions with mycotoxin [177] , [178] . Electron beam irradiation or UV irradiation induced more than 90 % degradation efficiency of ZEA, which may also be a new physical approach for mycotoxin elimination [179] , [180] . While, strong alkalis or oxidants including ammonia, hydrogen peroxide, sodium hypochlorite and ozone are used as chemical approaches to effectively break the structure of mycotoxin for detoxification [181] . Chemical experiments showed that ZEN could be degraded within 10 s after a high concentration of 2.0 mg/L ozone treatment [182] . Based on the adsorption and neutralization effects of microbial cell on mycotoxin, microbial cell can act as biological means for mycotoxin elimination [183] . For example, the supplementation of 1.2 × 10 11 CFU/Kg Bacillus licheniformis CK1 or 1 × 10 9 CFU/g Bacillus subtilis ANSB01G with feed products alleviated the toxic effects of ZEN in female piglets, suggesting that microorganisms or their enzymes could bio-transform mycotoxin [109] , [184] . Additionally, as shown in the Table 4 , multiple natural antioxidant compounds extracted from plants, including cyanidin-3-O-glucoside [172] , resveratrol [63] , [185] , saffron [186] , liquiritigenin [187] , scutellarin [188] , [189] , cinnamon [186] , vitamin C (ascorbic acid) [190] , chlorogenic acid [191] , grape seed extract [192] , proanthocyanidins (also known as condensed tannins) [193] , tannic acid (TA) [194] , quercetin [195] , xenoestrogens equol [196] , flavonoid compound isorhamnetin [197] , or animals-derived pineal gland-origin melatonin [52] , [198] , [199] , [200] , [201] , [202] , were able to alleviate the negative effects of mycotoxin in female mammal reproduction. These studies mostly focus on ameliorating ZEN-induced damage to GCs, cumulus oocyte complexes, parthenogenetic embryos and trophectoderm cells in swine through in vitro exposure. Little in vivo research has evaluated it at the individual level regarding reproductive organ injury, hormone secretion disorders and fetal development potential. Furthermore, it seems that the inhibition of oxidative stress-related mitochondria functions, DNA damage, cell cycle arrest, autophagy, and apoptosis through PI3K/AKT or MAPKs signaling pathways serves as the major protective mechanism against the toxic effects of mycotoxin. Cytoskeleton structure and epigenetic modification are also significantly ameliorated. However, since most studies co-treat mycotoxin with an effective compound, the possible biochemically interaction between the two compounds should be taken into account. The detoxification methods of other mycotoxins except ZEN should also be further confirmed especially through in vivo test. Table 4 Natural antioxidant compounds extracted from plants or animals for mycotoxin toxicities elimination/alleviation. Kinds Constituents Effective concentration and treated way Mycotoxin and concentration Treated cell type Mechanism Ref Plant-derived natural antioxidant Quercetin 100 ng/mL and 24 h in vitro co-incubation 100 ng/mL T-2 Porcine ovarian granulosa cell Increased TAS, SOD and GPx activity against oxidative stress [195] Cyanidin-3-O-glucoside 20 μM and 24 h in vitro co-incubation 30 μM ZEN Porcine ovarian granulosa cell Via the ITGA7-PI3K-AKT pathway against proliferation inhibition and apoptosis [172] Resveratrol Pretreated for 24 h in vitro 30 μM ZEN Human endometrial stromal cells Increased SIRT1, GPx, and PRL mRNA expression against oxidative stress in decidual cells [63] 2 μM and in vitro co-incubation 20 µM ZEN Porcine oocyte Enhance PINK1/Parkin-mediated mitophagy against mitochondrial dysfunction, oxidative stress and apoptosis [185] Liquiritigenin In vitro 10–40 μM 1 h preincubation, while 5 mg/Kg/day in vivo pre-injection 24 h in vitro 10 μM ochratoxin A, while 3 mg/Kg/day in vivo Mouse blastocyst Against mitochondria-related oxidative stress apoptosis [187] Scutellarin 500, 1000, 2000 μg/mL 24 h co-treated in vitro , while gavage 100 mg/Kg for 3 days in vivo 60 μM ZEN in vitro ; while gavage 40 mg/Kg for 2 h in vivo Mouse ovarian granulosa cells Against mitochondria-related cell cycle arrest and apoptosis via MAPK/JNK and heat shock protein-necroptosis pathway [188] , [189] Ascorbic acid (Vc) 50 μg/mL co-treated in vitro 1 nM AFB1 in vitro Yak oocyte and parthenogenetic embryos Against oxidative stress related mitochondrial dysfunction, apoptosis, as well as abnormal cytoskeleton and DNA methylation transferase level [190] Equol 1 μM co-treated in vitro for 3 days 1 μM ZEN in vitro for 3 days Ovine preantral follicles Against follicular degeneration and DNA damage [196] Silymarin 20 mg/Kg 20 mg/Kg ZEN for 6 weeks in vivo Rat Against ovary and uterus injury, as well as abnormal hormones synthesis and ABC transporters [110] Grape seed proanthocyanidin 200 μM co-treated in vitro for 44 h 30 μM FB1 in vitro for 44 h Porcine cumulus oocyte complexes Against mitochondrial dysfunction and oxidative stress related apoptosis and autophagy, as well as cell abnormal cycle progression and cytoskeletal structure [193] Grape seed extract 150 mg/Kg BW throughout the gestation period in vivo 25 mg/Kg BW ZEN during 6–13 of gestation in vivo Pregnant mice Against genotoxicity and developmental toxicity in fetus [192] Isorhamnetin 10 μM co-treated for 44 h in vitro 5 μM ZEN for 44 h in vitro Porcine cumulus oocyte complexes Against oxidative stress related mitochondrial dysfunction and apoptosis, as well as ER stress through PI3K/AKT signaling pathway [197] Tannic acid 200 mg/Kg BW co-treated for 7 days in vivo 10 mg/Kg BW ZEN for 7 days in vivo Mice Against reproductive hormones secretion disorders and oxidative damage by regulation on death receptor and mitochondrial apoptosis signaling pathway in ovarian tissue [194] Chlorogenic acid 250 μg/mL co-treated in vitro 60 μM ZEN in vitro Mouse ovarian granulosa cells Against apoptosis [191] Animal-derived natural antioxidant Melatonin 1 mM co-treated in vitro 10 μM AFB1 in vitro Porcine oocytes and parthenogenetic embryos Against mitochondria related oxidative stress and apoptosis in oocyte and cytochrome P450 system activity in cumulus cells [52] 10 μM in vitro co-treated, while co-injection 30 mg/Kg BW for 7 days in vivo 2 μM DON in vitro , while 0.6 mg/Kg BW DON per day for 7 days Mouse oocyte and early embryo Against oxidative stress-mediated apoptosis, acetylated tubulin-related spindle formation, K-MT attachment and aneuploidy, as well as DNA and histone methylation [202] 1 μM in vitro co-treated 2 μM DON in vitro Murine ovary granulosa cells Against oxidative stress, mitochondrial dysfunction and inflammation related apoptosis, as well as reproduction hormone gene expression by regulation on NF-kB and MAPKs activity [201] 10 μM co-treated in vitro 8 μM OTA in vitro Porcine oocyte Against mitochondria dysfunction and oxidative stress-related apoptosis/autophagy, as well as spindle formation and cell cycle progression failure [200] 1 μM co-treated in vitro 5 μM ZEN in vitro Porcine parthenogenetic embryos Against oxidative stress related mitochondrial dysfunction, DNA damage, apoptosis, and autophagy [198] Pretreated 100 μM for 12 h in vitro 25 μM β-zearalenol (β-zol) or 50 nM HT-2 toxin for 24 h in vitro Bovine ovarian granulosa cells Against oxidative stress and apoptosis by regulation on p38-MAPK [19] Natural antioxidant compounds extracted from plants or animals for mycotoxin toxicities elimination/alleviation. Although various methods have been tried to reduce the exposure dose of mycotoxin or its adverse effects in food, feed, individual or cell as much as possible, the main issue of decreased quality and nutritional compositions in the food by chemical oxidation and physical absorption remains unresolved. Besides, slow progression of biological breeding and difficulty in reutilizing bacterial agents on a large scale limit their extensive use in production practices. Effective and available ways to eliminate or alleviate mycotoxin toxicities are still urgent to be settled.

25 % of the crops in the world are contaminated by mycotoxin, leading to nearly 1 billion tons of feed/food losses and 930 million USD economic costs per year [1] , [2] . Mycotoxins are secondary metabolites produced by fungi and about 400 mycotoxins have been recognized. Among them, aflatoxins (AFs, such as aflatoxins B1 (AFB1)); zearalenone (ZEN); ochratoxin A (OTA); fumonisins B (FBs, such as fumonisin B1 (FB1)); type A trichothecenes such as T-2 toxin and HT-2 toxin, and type B trichothecenes such as deoxynivalenol (DON) and nivalenol (NIV) are the most relevant and typical groups of mycotoxins found in food [3] . Mycotoxin contamination commonly exists in cereals, fruits, milk, and vegetables, etc. [4] . For dietary exposure, the estimated daily intakes of FBs and OTA were 55–237 ng/Kg/BW/day and 0.65–5.72 ng/Kg/BW/day in China during 2007–2020 respectively, which were above the upper-bound values of 2.77-40.8 % of the provisional tolerable weekly intake [5] . While for feedstuffs, a total of 1569 samples collected from China between 2016 and 2017 showed that the individual occurrence rates of AFB1, ZEN, and DON were all more than 70 % [6] . More recent detailed data on mycotoxin occurrence in the foods, feeds and their products among different countries were shown in the Table 1 [5] , [6] , [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] , implying a widespread occurrence of mycotoxins and their potential toxicities to animals and humans. Table 1 Mycotoxin exposure in cereal, food or feed among different countries. Mycotoxin Country Occurrence (foods or feeds) Incidence (%) Range or mean ( μ g/Kg) Ref Aflatoxins China Maize 94.9 Up to 67.6 [6] China Maize bran 100 Up to 13.5 [6] North America Corn 26 Up to 920 a [7] Sorth America Corn 25 Up to 273 a [7] Central Europe Corn 31 Up to 3 a [7] Southern Europe Corn 36 Up to 44 a [7] Pakistan Rice and rice products 35 Up to 32.2 [8] Serbia Maize 90 0.4–41 [9] Zearalenone China Maize 93.4 Up to 624.3 [6] China Maize bran 100 Up to 1268.6 [6] Brazil Wheat 12 22.5–133 [10] Brazil Wheat 84 20.4–233 [10] South Korean Compound feeds 96.4 1–932 [11] South Korean Feed ingredients 77 1–1330 [11] Netherlands Wheat flour 100 12.4–13.7 [12] Netherlands Mixed-flour 50 19.8–37.2 [12] Portugal Wheat flour 23.5 7.4–15.3 [12] Portugal Mixed-flour 30.8 5.4–39.4 [12] Switzerland Wheat 32 NA-3070 [13] Serbia Winter wheat 94.6 16–201 [14] North America Corn 29 Up to 4787 a [7] Sorth America Corn 43 Up to 1800 a [7] Central Europe Corn 39 Up to 849 a [7] Southern Europe Corn 21 Up to 1546 a [7] Serbia Maize 53 1–58 [9] Ochratoxin A North America Corn 10 Up to 18 a [7] Sorth America Corn 12 Up to 355 a [7] Central Europe Corn 10 Up to 3 a [7] Southern Europe Corn 29 Up to 46 a [7] China 12 food categories b 4.9 Up to 6.69 [5] Belgian Foodstuff c 46 Up to 10.29 [15] Moroccan Bread 81.8 0.11–5.04 [16] Moroccan Semolina 74.5 0.15–4.01 [16] Moroccan Pasta 75.2 0.10–6.77 [16] Pakistan Rice and rice products 19 Up to 24.9 [8] Fumonisins B Argentina Wheat-based products 74 Up to 18.9 [17] Serbia Winter wheat 92 750–4900 [14] North America Corn 39 Up to 22900 a [7] Sorth America Corn 92 Up to 53700 a [7] Central Europe Corn 60 Up to 7680 a [7] Southern Europe Corn 90 Up to 11050 a [7] China Corn based products 98.1 < 0.27–5046 [18] China Wheat flour 6.2 0.3–34.6 [18] China 12 food categories b 32.6 Up to 38.0 [5] Trichothecenes China Maize 98.4 Up to 4590.8 [6] China Maize bran 100 Up to 4710.7 [6] Brazil Wheat 96 200–862 [10] Brazil Wheat 100 495–2150 [10] Switzerland Wheat 80 NA-10600 [13] Switzerland Wheat 21 NA-470 [13] Serbia Winter wheat 60 86–200 [14] Serbia Winter wheat 93.3 50–1090 [14] North America Corn 79 Up to 24900 a [7] Sorth America Corn 17 Up to 939 a [7] Central Europe Corn 72 Up to 26121 a [7] Southern Europe Corn 47 Up to 3851 a [7] a: ppb. b: 12 food categories include cereals, legumes, potatoes, meats, eggs, aquatic products, dairy products, vegetables, fruits, sugars, beverages, and alcohols. c: foodstuffs include cereal-based products, fruit and vegetable juices, dried fruit, nuts, seeds, herbs, spices, meat imitates, alcoholic beverages, baby food products, coffee and food supplements. Mycotoxin exposure in cereal, food or feed among different countries. a: ppb. b: 12 food categories include cereals, legumes, potatoes, meats, eggs, aquatic products, dairy products, vegetables, fruits, sugars, beverages, and alcohols. c: foodstuffs include cereal-based products, fruit and vegetable juices, dried fruit, nuts, seeds, herbs, spices, meat imitates, alcoholic beverages, baby food products, coffee and food supplements. Acute and chronic animal tests showed that mycotoxin exposure induced abdominal pain, vomiting, diarrhea, depression, anorexia, growth retardation and multifocal organs damage such as cellular carcinoma, enteritis, pulmonary or cerebral oedema, hepatitis, and nephrosis [3] , [19] , [20] . Aberrant vulva area, myometrium and endometrium hypertrophy were observed in the post-weaning gilts or rats after ZEN exposure [21] , [22] , [23] . Moreover, DNA adducts were formed with ZEN in genitalia of mice and rats [24] , indicating its possible role in reproductive organ cancer. OTA in vivo exposure led to pup loss and delayed puberty of F1 rats [25] , while skeletal and visceral developmental abnormalities of fetus were seen in animals administered DON, AFB1 and OTA [20] , [26] . These results show the diverse and powerful toxic effects of mycotoxin on health, such as neurotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, and reproductive toxicity. Moreover, multiple human exposure assessments revealed that at least two or more mycotoxins were found in urine and serum. Among them, ZEN, AFs, FBs, OTA, and type B trichothecenes were the most prevalent which were nearly 50 % detection, some even exceeded the tolerable daily intake [27] , [28] . More importantly, 73 % of human amniotic fluid samples from fetus with genetic defects were detected with mycotoxins, with the presence rates of NIV, AFs, OTA and DON being 33.7 %, 31.4 %, 26.7 %, and 27.9 % [29] . Low birth weight [30] , [31] , [32] , neonatal jaundice [30] , [33] , eclampsia [34] , preterm birth [35] , and fetal growth faltering [32] , [36] were observed when AFs, OTA, FB1, and DON exposure to prenatal women. These findings indicate that there is significant reproductive toxicity of mycotoxin and huge potential risks of mycotoxin exposure to fetus development and newborn health. The mycotoxin formation depends on the fungi species and environmental factors such as temperature, moisture, PH, and substrate composition [37] . Warm and wet climate, together with poor food quality and crop storage conditions promote mycotoxin formation [4] . Moreover, characteristics of mycotoxin, such as high melting point and solubility, make it hard to remove by traditional processing approaches such as cooking, baking, roasting, and pasteurization [38] , [39] , [40] . The wide production conditions and strong physical properties of mycotoxin result in an unpredictable and unavoidable problem of mycotoxin contamination, which is of great importance to continuously focusing on the studies about its exposure, toxicity and specific mechanisms, together with effective controlling methods.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.